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Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen
associated with many types of chronic infection. At sites of chronic infection,
such as the airways of people with cystic fibrosis (CF), P.
aeruginosa forms biofilm-like aggregates. These are clusters of
bacterial cells encased in a polymer-rich matrix that shields bacteria from
environmental stresses and antibiotic treatment. When P.
aeruginosa forms a biofilm, large amounts of filamentous Pf
bacteriophage (phage) are produced. Unlike most phage that typically lyse and
kill their bacterial hosts, filamentous phage of the genus Inovirus, which
includes Pf phage, often do not, and instead are continuously extruded from the
bacteria. Here, we discuss the implications of the accumulation of filamentous
Pf phage in the biofilm matrix, where they interact with matrix polymers to
organize the biofilm into a highly ordered liquid crystal. This structural
configuration promotes bacterial adhesion, desiccation survival, and antibiotic
tolerance – all features typically associated with biofilms. We propose that Pf
phage make structural contributions to P. aeruginosa biofilms
and that this constitutes a novel form of symbiosis between bacteria and
bacteriophage.

Filamentous bacteriophage in P. aeruginosa biofilms

When P. aeruginosa forms a biofilm, Pf phages are produced in
abundance. This has been observed by several laboratories using different methods to
grow biofilms (flow cells, chemostats, etc.). In addition to being produced under
biofilm conditions in vitro, Pf prophages are also prevalent
amongst clinical P. aeruginosa CF isolates. Indeed, we were able to
detect intact Pf phage in CF sputum at concentrations averaging
107/ml.

Filamentous bacteriophage and liquid crystal assembly

It is well established that filamentous Inoviruses, including Pf phage, can assemble
liquid crystals – a state of matter between that of a liquid and a solid. Indeed,
due to their uniform (monodisperse) diameter and length, Inoviruses have been used
for years as an experimental model system to study the physics of liquid crystals.
High concentrations of filamentous phage will spontaneously align and form a liquid
crystal, owing to steric forces between neighboring phage particles. Non-adsorbing
polymers can potentiate this liquid crystal assembly through a crowding mechanism
called depletion attraction. This is an entropic force that causes the phage to
segregate from the polymers, resulting in an enhanced propensity to form liquid
crystals (Figure 1).

Figure 1

(A) Dashed lines around phage particles represent an exclusion
volume not accessible to the polymer coil’s center of mass. When phages are
dispersed, the volume inaccessible to the polymers is maximized.

(B) When phages come into close proximity to each other, their
exclusion volumes overlap, increasing the total volume accessible to the
polymers. This maximizes the entropy of the system. Arrows represent an
osmotic imbalance that polymers exert on the liquid crystalline phage
bundle. This osmotic pressure is what physically holds the liquid crystal
together.

(D) As time progresses, the phages form liquid crystalline
structures.

The biophysical properties of Pf phage transform the biofilm matrix into a liquid
crystal

Given its inherently crowded, polymer-rich nature, the biofilm matrix offers an ideal
environment for depletion attraction to organize filamentous phage into liquid
crystalline structures. We quantified the liquid crystalline properties of
P. aeruginosa biofilms by taking advantage of the optical
properties of liquid crystals. In particular, they exhibit birefringence, or double
refraction, whereby incoming light is split into two beams with perpendicular
polarization. Thus, birefringence is a direct measurement of molecular order. Using
this technique in conjunction with studies that included Pf phage deficient and
replete P. aeruginosa strains, we found that Pf phage were both
necessary and sufficient to assemble P. aeruginosa biofilms into
birefringent liquid crystals (Figure 2).

Figure 2

The birefringence of the liquid crystalline biofilm matrix can be visualized
by placing colony biofilms on an agar pad between crossed polarizing lenses.
The liquid crystalline matrix can change the polarization of light allowing
it to pass through both polarizing lenses. Thus, non-liquid crystalline
biofilms appear opaque (red arrow) while liquid crystalline biofilms appear
bright (blue arrow). The organization of the biofilm matrix into a liquid
crystal enhances adhesion, antibiotic tolerance, and desiccation survival.
Further, Pf phage can increase the viscosity of host polymers such as mucin
and DNA.

Pf phage and a liquid crystalline matrix enhance the pathogenic features of
P. aeruginosa biofilms

The crystalline structure of the matrix enhanced biofilm fitness in several ways. One
function of the biofilm matrix is to offer bacteria protection from desiccation. We
found that bacteria within a liquid crystalline matrix more efficiently retained
moisture and were better able to survive desiccation than bacteria within an
unordered matrix. This enhanced ability to withstand desiccation may be due to the
inherent viscosity of liquid crystals, as viscous materials generally display low
rates of evaporation. It is interesting to note that epidemic strains of P.
aeruginosa, such as the Liverpool epidemic strain, contain Pf prophage.
Given that desiccation tolerance is important for the transmission of bacteria from
the environment to a host, Pf phage may enhance the desiccation survival of epidemic
strains, contributing to their transmissibility.

We also found that a crystalline biofilm matrix protects bacteria from killing by
antibiotics. Bacteria within a liquid crystalline matrix were more tolerant to
aminoglycosides such as tobramycin. Aminoglycosides are cationic antibiotics;
therefore we hypothesized that they would be sequestered by anionic Pf phage in the
biofilm matrix, reducing bacterial killing. This was indeed the case. Moreover,
liquid crystalline phases of Pf phage were able to bind tobramycin more efficiently
than disordered Pf phage. While the mechanism behind this enhanced binding is still
unclear, we speculate that bundles of negatively charged Pf phage within the liquid
crystalline biofilm matrix would create a dense, localized negative charge that
could attract and sequester charged antibiotics. One prediction of this model is
that antibiotics with a net neutral charge, like ciprofloxacin, would not be
sequestered by Pf phage. Indeed, bacteria within a crystalline matrix were equally
sensitive to ciprofloxacin relative to bacteria in a non-crystalline matrix.
Together, these data support a role for Pf phage in tolerance to aminoglycosides and
potentially other cationic antimicrobials, such as antimicrobial peptides.

Host polymers and Pf bacteriophage

In CF, viscous airway secretions containing mucin, DNA, F-actin, hyaluronan, and
other polymers accumulate in the airways, contributing to pathogenesis. We found
that in addition to bacterial polymers, Pf phage likewise assemble liquid crystals
in the presence of host polymers, causing an increase in viscosity. This ability of
Pf phage to increase the viscosity of the local environment could trap bacteria at
sites of infection, reducing bacterial invasiveness, thereby promoting a chronic,
localized infection.

Conclusions

Our findings highlight a previously unknown role for filamentous Pf phage in
organizing the P. aeruginosa biofilm matrix into a liquid
crystalline structure. These findings help ground our understanding of biofilm
formation within established paradigms of soft matter physics.

We demonstrated that Pf phage contribute to several bacterial phenotypes related to
chronic infection as a result of these structural contributions to the P.
aeruginosa biofilm matrix (Figure 2). In the context of CF, Pf phage
may allow P. aeruginosa to resist desiccation outside of the body,
which could potentially increase the chances that it can be transmitted to a host
and establish chronic airway infections. Once P. aeruginosa is
established within the airways of infected individuals, Pf phage may increase the
viscosity of CF airway secretions, and potentially contribute to bacterial
colonization and airway obstruction. As the infection becomes further established,
the liquid crystalline biofilm matrix could enhance antimicrobial tolerance, further
promoting infection persistence.

In light of these potential contributions to chronic P. aeruginosa
infection, targeting the production of filamentous phage or the liquid crystalline
organization of the biofilm matrix may offer alternative therapeutic approaches.
Conceivably, these could be directed at disrupting biofilm assembly, dispersing
pre-existing biofilms, or increasing the susceptibility of bacteria to
antimicrobials.

It is tempting to speculate that filamentous phage may likewise organize biofilms
formed by other bacterial species into liquid crystalline structures. Inoviruses are
prevalent amongst Gram-negative bacterial species (e.g., Escherichia coli,
Vibrio cholerae, etc.) and we showed that some of these other phages
likewise assemble liquid crystals. Further, in addition to filamentous phage, phage
with other capsid geometries, such as those with an icosahedral head, might also
influence matrix structure and function when produced in abundance. For example,
depletion attraction can cause spherical particles to aggregate and form a variety
of structures that link together into extended networks (i.e., they form a gel).

Finally, we propose that Pf phage may play a more symbiotic role in P.
aeruginosa biofilms than previously suspected. The production of Pf
phage has been linked to the progression of the biofilm lifecycle by promoting
localized cell lysis, resulting in the accumulation of substrate (e.g. DNA) for
biofilm formation. While Pf phage only induces lysis in a subpopulation of bacterial
cells, our findings suggest that it may also contribute to bacterial fitness through
effects on adhesion, viscosity, tolerance to desiccation, and tolerance to
antibiotics.

Funding Statement

This work was supported by National Institutes of Health grants
HL098067, HL089455, AI101307, and HL113294; the Microbiology Core of the NIDDK
Cystic Fibrosis Research and Translation Center (DK089507); and a Cystic
Fibrosis Foundation Research Development Program Postdoctoral Fellowship to
P.R.S.